Background The aim of this thesis was to develop an LC-MS / MS method for the individuation and dosage of THCCOOH, the main THC metabolite, and THC-A and CBD-A, inactive precursors of the THC and CBD cannabinoids. , in urine samples, taken from subjects treated by the addiction services of the province of Pavia (SerD). Methods Initially urine samples were divided into two aliquots. The first aliquot was analyzed as such, while the second aliquot was subjected to hydrolysis with NaOH (5M). The urine samples were then directly injected into the LC-MS / MS system, following a simple dilution 1:20 in bidistilled water, containing THCCOOH-D3 as an internal standard. The analytes were separated on column C18 working in reverse phase, and detected with a triple quadrupole tandem mass spectrometer operating in MRM mode and negative polarization. Two ionic transitions were selected for each analyte. The method has been submitted to complete validation ,following the dictates of the main international guidelines. The analytical procedure was then applied to 47 samples of urine taken from subjects who had declared a recent use of preparations based on inhaled cannabis plant. Results and Discussion The method developed was simple and fast and all the validation parameters fall whitin the acceptability limits, according to the aforementioned international guidelines. 43 of the 47 samples tested were positive for THCCOOH, confirming the intake of cannabis plant derivatives by the test subjects. Furthermore, three of the 43 THC-positive subjects were also positive at THC-A. Four subjects had declared only the consumption of "legal" preparations of cannabis plant (products containing a concentration of THC less than 0.2%, and of CBD above 15%). Three out of four of the users of "legal cannabis" tested positive for CBD-A, while all four were negative for THCCOOH and THC-A.
Background Lo scopo di questo elaborato di tesi è stato quello di sviluppare una metodica LC-MS/MS per la ricerca e il dosaggio di THCCOOH, principale metabolita del THC, e di THC-A e CBD-A, precursori inattivi dei cannabinoidi THC e CBD, in campioni di urina, prelevati a soggetti in cura presso i servizi delle dipendenze della provincia di Pavia (SerD). Metodi I campioni di urina sono stati inizialmente suddivisi in due aliquote. La prima aliquota è stata analizzata tal quale, mentre la seconda aliquota è stata sottoposta ad idrolizzazione con NaOH (5M). I campioni di urina sono stati quindi direttamente iniettati nel sistema LC-MS/MS a seguito di una semplice diluizione 1:20 in acqua bidistillata, contenente THCCOOH-D3 quale standard interno. Gli analiti sono stati separati su colonna C18 operante in fase inversa, e rilevati con spettrometro di massa tandem a triplo quadrupolo operante in modalità MRM e polarità negativa. Sono state selezionate due transizioni ioniche per ciascun analita. Il metodo è stato sottoposto a completa validazione, seguendo i dettami delle principali linee guida internazionali. La procedura analitica è stata quindi applicata a 47 campioni di urina prelevati a soggetti che avevano dichiarato un uso recente di preparazioni a base di pianta cannabis per via inalatoria. Risultati e Discussione La metodica sviluppata è risultata semplice e veloce e tutti i parametri di validazione sono risultati rientrare nelle soglie di accettabilità secondo le suddette linee guida internazionali. 43 dei 47 campioni analizzati sono risultati positivi al THCCOOH, confermando l’assunzione di derivati della pianta cannabis da parte dei soggetti in esame. Inoltre tre dei 43 soggetti positivi al THC sono risultati positivi anche al THC-A. 4 soggetti avevano dichiarato il solo consumo di preparazioni “legali” di pianta cannabis (prodotti contenenti una concentrazione di THC inferiore a 0.2%, e di CBD superiore al 15%). Tre su quattro dei soggetti utilizzatori di “cannabis legale” sono risultati positivi al CBD-A, mentre tutti e 4 sono risultati negativi per THCCOOH e THC-A.
RICERCA E DOSAGGIO DI THCCOOH, THC-A E CBD-A IN CAMPIONI DI URINA MEDIANTE LC-MS/MS
CADILE, FRANCESCA
2017/2018
Abstract
Background The aim of this thesis was to develop an LC-MS / MS method for the individuation and dosage of THCCOOH, the main THC metabolite, and THC-A and CBD-A, inactive precursors of the THC and CBD cannabinoids. , in urine samples, taken from subjects treated by the addiction services of the province of Pavia (SerD). Methods Initially urine samples were divided into two aliquots. The first aliquot was analyzed as such, while the second aliquot was subjected to hydrolysis with NaOH (5M). The urine samples were then directly injected into the LC-MS / MS system, following a simple dilution 1:20 in bidistilled water, containing THCCOOH-D3 as an internal standard. The analytes were separated on column C18 working in reverse phase, and detected with a triple quadrupole tandem mass spectrometer operating in MRM mode and negative polarization. Two ionic transitions were selected for each analyte. The method has been submitted to complete validation ,following the dictates of the main international guidelines. The analytical procedure was then applied to 47 samples of urine taken from subjects who had declared a recent use of preparations based on inhaled cannabis plant. Results and Discussion The method developed was simple and fast and all the validation parameters fall whitin the acceptability limits, according to the aforementioned international guidelines. 43 of the 47 samples tested were positive for THCCOOH, confirming the intake of cannabis plant derivatives by the test subjects. Furthermore, three of the 43 THC-positive subjects were also positive at THC-A. Four subjects had declared only the consumption of "legal" preparations of cannabis plant (products containing a concentration of THC less than 0.2%, and of CBD above 15%). Three out of four of the users of "legal cannabis" tested positive for CBD-A, while all four were negative for THCCOOH and THC-A.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
Per maggiori informazioni e per verifiche sull'eventuale disponibilità del file scrivere a: unitesi@unipv.it.
https://hdl.handle.net/20.500.14239/23735